PCRPolymerase Chain

Reaction

Mariam Cortes Tormo

Miami Children’s Hospital

Research institute

2013

Amplification reaction

Polymerase Chain Reaction (PCR) is done in three steps that constitute a cycle, repeated for a certain amount of times:

1 - Denaturation2 - Hybridization3 - ElongationThe time, temperature and number of cycles are factors

that determin the results of the PCR, thus by modifying we can optimize the reaction

Denaturation

This is a critical step as it is very important that the DNA template be completely denatured

• It is recommended temperatures of 94°-95° C, for 30'' to1‘• High G + C increases the time or temperature• The enzyme activity decreases very rapidly to from 95 ° C. At

these temperatures or higher is recommended to decrease the incubation time

.

In practice, before start cycles, is normal to add a denaturation period

This step is usually 94°C 5'

Hybridization

Temperature and time will depend on 3 factors related to the founders:

• Composition of bases

• Size

• Concentration

In practice, the annealing temperature may range between 45°C and 65°C, for a time ranging from 30” to 1’

An increase in temperature promotes the specificity; where as a decrease will promote incorrect connections of the initiators

Increased time promotes less specificity fornonspecific amplification products

Elongation

In most reactions, the extension step performed at 72 ° C.

This temperature can range between 70-72°CThe extension time depends on the size of the amplification. You can estimate about 1 minute to 1 kb elongatedIn practice it is normal for the end of all cycles to perform an ultimate elongation of 72°C from 5'

Amplified DNA

Cycles

Some things to know…

Not using a high number of cycles (25-45). The error rate is proportional to the number of cycles

The concentration of dNTPs must be equal for the 4 types, while being as low as possible without losing performance

Reduce the time as much as possible in each step The concentration of Mg ++ should not be excessive.

This decreases the specificity of PCR

Dimethyl sulfoxide (DMSO 10%) contributes todecreasing the secondary structure of DNA

Buffers which help to stabilize the enzyme

...to get the best conditions to reduce errors:

Between 20-200 mMol

The concentration of Mg ++ must be 0.5 - 1.0 mM times the concentration of dNTPs

How many PCR’ s can we find?

Nested PCR In situ PCR Multiplex PCR RT-PCR Real Time PCR Random primer PCR

Nested PCR

Nested primers are used to increase the specificity of an amplification or to reamplify the product of a PCR reaction that did not yield enough material

Purpuse : Diagnosis of infectious diseases

Highly specific

In situ PCR

Mainly used in two aspects• Detection of exogenous gene fragment, improve the detection rate of virus infection,

concentrated in the inspection, such as HIV, HPV, HBV, CMV, etc • To observe the distribution of pathogens in the body • Endogenous gene fragments, such as human monogenic disease, gene

recombination, translocation of chromosomes, Ig fragments of mRNA gene fragments, etc

• Detection of gene transfection

Multiplex PCR

Multiplex polymerase chain reaction (PCR) is a variant of PCR in which two or more loci are simultaneously amplified in the same reaction

Since its first description in 1988, this method has been successfully applied in many areas of DNA testing, including analyses of deletions, mutations and polymorphisms or quantitative assays and reversetranscription PCR

Some of the applications of multiplex PCR include:

• Pathogen Identification• High Throughput SNP Genotyping• Mutation Analysis• Gene Deletion Analysis• Template Quantitation• Linkage Analysis• RNA Detection• Forensic Studies• Diet Analysis

RT-PCR

This technique is commonly used in molecular biology to detect RNA expression levels

It is used to qualitatively detect gene expression through creation of complementary DNA (cDNA) transcripts from RNA

• Genetic Disease Diagnosis :RT-PCR can be used to diagnose genetic disease such as Lesch–Nyhan syndrome. This genetic disease is caused by a malfunction in the HPRT1 gene, which clinically leads to the fatal uric acid urinary stone and symptoms similar to gout. Analyzing a pregnant mother and a fetus for mRNA expression levels of HPRT1 will reveal if the mother is a carrier and if the fetus will likely to develop Lesch–Nyhan syndrome• Cancer Detection : Scientists are working on ways to use RT-PCR in cancer detection to help improve prognosis, and monitor response to therapy. Circulating tumor cells produce unique mRNA transcripts depending on the type of cancer. • RT-PCR is commonly used in studying the genomes of viruses whose genomes are composed of RNA, such as Influenzavirus A and retroviruses like HIV• RT-PCR can also be very useful in the insertion of eukaryotic genes into prokaryotes

Real Time PCRIs based on the polymerase chain reaction (PCR), which is used to amplify and simultaneously quantify a targeted DNA molecule

Random primer PCRTECHNIQUE USED PRIMES CONSISTING OF A POOL OF SEQUENCE ENDS 3 'REGION OF DNA AND A 5' CONSTANT FOR THE DETECTION OF FIRST BUILT

APPLICATION ALLOWS THE AMPLIFICATION OF DNA MOLECULES AND GENOME THAT VARY FROM 400 TO 40 PAIRS OF MEGA BASES

THIS VERSION OF TECHNICAL SPECIFICATIONS IS VERY SENSITIVE AND IS VERY USEFUL IN THE STUDIO AND DIAGNOSIS OF BODIES OF THE SAME KIND WITH SPECIFIC GENETIC VARIATIONS

Thank you very much

for your attention